MAPK信号通路调节卷烟烟气诱导的炎症因子的释放
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摘要
为评估卷烟烟气对人支气管上皮细胞(BEAS-2B)的毒性效应,使用细胞计数试剂盒(CCK-8)对BEAS-2B细胞进行检测,使用光学显微镜对细胞形态进行观察,采用Western Blot实验检测丝裂原活化蛋白激酶(MAPK)家族的细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基末端激酶1/2(JNK1/2)和p38的磷酸化水平的变化,采用酶联免疫吸附实验检测炎症因子白介素-1β(IL-1β)、IL-6和IL-8的释放水平。结果表明:①卷烟烟气染毒会显著降低BEAS-2B细胞的存活率。②卷烟烟气染毒会激活磷酸化的ERK1/2、JNK1/2和p38信号通路,并且会诱导炎症因子IL-1β、IL-6和IL-8释放水平升高。③特异性抑制剂分别抑制ERK1/2、JNK1/2和p38信号通路后,炎症因子释放水平显著下降。卷烟烟气诱导的炎症受MAPK信号通路的调节,该结果可为卷烟烟气诱导的肺部炎症相关疾病的机制研究提供参考。
To evaluate the toxic effects of cigarette smoke on human bronchial epithelial cells(BEAS-2 B cells),the viability of BEAS-2 B cells was detected with a cell counting kit(CCK-8) and the cell morphology was observed under an optical microscopy. Western Blot assay was conducted to detect the expression variations of extracellular-regulated protein kinases 1/2(ERK1/2) and c-Jun N-terminal kinase(JNK1/2) and the phosphorylation level of p38. The release levels of inflammatory cytokines interleukin-(IL-)1β, IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay. The results showed that: 1) The viability of BEAS-2 B cells significantly decreased after exposure to cigarette smoke. 2) Exposure to cigarette smoke activated the expression of phospho-(P-)ERK1/2, P-JNK1/2, P-p38 pathways and induced the release of inflammatory cytokines IL-1β, IL-6 and IL-8. 3) The release levels of inflammatory cytokines decreased significantly after ERK1/2, JNK1/2 and p38 pathways' being inhibited by specific inhibitors. In conclusion, the inflammation induced by cigarette smoke is regulated by mitogen-activated protein kinase(MAPK) pathways. These results provide a reference for the mechanism analysis of pulmonary inflammatory diseases induced by cigarette smoke.
To evaluate the toxic effects of cigarette smoke on human bronchial epithelial cells(BEAS-2 B cells),the viability of BEAS-2 B cells was detected with a cell counting kit(CCK-8) and the cell morphology was observed under an optical microscopy. Western Blot assay was conducted to detect the expression variations of extracellular-regulated protein kinases 1/2(ERK1/2) and c-Jun N-terminal kinase(JNK1/2) and the phosphorylation level of p38. The release levels of inflammatory cytokines interleukin-(IL-)1β, IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay. The results showed that: 1) The viability of BEAS-2 B cells significantly decreased after exposure to cigarette smoke. 2) Exposure to cigarette smoke activated the expression of phospho-(P-)ERK1/2, P-JNK1/2, P-p38 pathways and induced the release of inflammatory cytokines IL-1β, IL-6 and IL-8. 3) The release levels of inflammatory cytokines decreased significantly after ERK1/2, JNK1/2 and p38 pathways' being inhibited by specific inhibitors. In conclusion, the inflammation induced by cigarette smoke is regulated by mitogen-activated protein kinase(MAPK) pathways. These results provide a reference for the mechanism analysis of pulmonary inflammatory diseases induced by cigarette smoke.
引文
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[4]YUAN Jin,LIU Renping,MA Yaohui,et al.Curcumin attenuates airway inflammation and airway remolding by inhibiting NF-kappa B signaling and COX-2 in cigarette smoke-induced COPD mice[J].Inflammation,2018,41(5):1804-1814.
[5]Kim C W,Go R E,Hwang K A,et al.Effects of cigarette smoke extracts on apoptosis and oxidative stress in two models of ovarian cancer in vitro[J].Toxicology in Vitro,2018,52:161-169.
[6]CHEN Zhihai,WANG Dapeng,LIU Xing,et al.Oxidative DNA damage is involved in cigarette smokeinduced lung injury in rats[J].Environmental Health and Preventive Medicine,2015,20(5):318-324.
[7]WANG Limeng,LI Xiang,YANG Zhihua,et al.Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K,AMPKand MAPK pathways[J].Environmental Pollution,2017,228:287-296.
[8]Turner M D,Nedjai B,Hurst T,et al.Cytokines and chemokines:At the crossroads of cell signalling and inflammatory disease[J].Biochimica et Biophysica Acta,2014,1843(11):2563-2582.
[9]DONG Ran,XIE Liang,ZHAO Kaishun,et al.Cigarette smoke-induced lung inflammation in COPDmediated via LTB4/BLT1/SOCS1 pathway[J].International Journal of Chronic Obstructive Pulmonary Disease,2016,11:31-41.
[10]SUI Xinbing,KONG Na,YE Li,et al.p38 and JNKMAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents[J].Cancer Letters,2014,344(2):174-179.
[11]SHEN Haitao,WU Na,WANG Yu,et al.JNK inhibitor SP600125 attenuates paraquat-induced acute lung injury:an in vivo and in vitro study[J].Inflammation,2017,40(4):1319-1330.
[12]HE Miao,Ichinose T,Yoshida S,et al.PM2.5-induced lung inflammation in mice:Differences of inflammatory response in macrophages and type IIalveolar cells[J].Journal of Applied Toxicology,2017,37(10):1203-1218.
[13]Mercer B A,Kolesnikova N,Sonett J,et al.Extracellular regulated kinase/mitogen activated protein kinase is up-regulated in pulmonary emphysema and mediates matrix metalloproteinase-1 induction by cigarette smoke[J].The Journal of Biological Chemistry,2004,279(17):17690-17696.
[14]LI Chunlei,YAN Yue,SHI Qi,et al.Recuperating lung decoction attenuates inflammation and oxidation in cigarette smoke-induced COPD in rats via activation of ERK and Nrf2 pathways[J].Cell Biochemistry and Function,2017,35(5):278-286.
[15]ISO 4387:2000 Cigarettes-Determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine[S].
[16]Kim E K,Choi E J.Compromised MAPK signaling in human diseases:an update[J].Archives of Toxicology,2015,89(6):867-882.
[17]Borthwick L A.The IL-1 cytokine family and its role in inflammation and fibrosis in the lung[J].Seminars in Immunopathology,2016,38(4):517-534.
[18]Arthur J S,Ley S C.Mitogen-activated protein kinases in innate immunity[J].Nature Reviews:Immunology,2013,13(9):679-692.
[19]Shin N R,Kim S H,Ko J W,et al.HemoHIM,a herbal preparation,alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide[J].Laboratory Animal Research,2017,33(1):40-47.
[20]Dumitru C D,Ceci J D,Tsatsanis C,et al.TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway[J].Cell,2000,103(7):1071-1083.
[21]Kaiser F,Cook D,Papoutsopoulou S,et al.TPL-2negatively regulates interferon-beta production in macrophages and myeloid dendritic cells[J].The Journal of Experimental Medicine,2009,206(9):1863-1871.
[22]Martinez F O,Helming L,Gordon S.Alternative activation of macrophages:an immunologic functional perspective[J].Annual Review of Immunology,2009,27:451-483.
[23]TIAN Yange,LI Ya,LI Jiansheng,et al.Bufei yishen granules combined with acupoint sticking therapy suppress inflammation in chronic obstructive pulmonary disease rats:via JNK/p38 signaling pathway[J].Evidence-Based Complementary and Alternative Medicine,2017:1-10.(2017-10-30)[2018-11-30].https://doi.org/10.1155/2017/1768243.
[24]Kang Y J,Bang B R,Otsuka M,et al.Tissue-specific regulation of p38α-mediated inflammation in Con A-induced acute liver damage[J].Journal of Immunology,2015,194(10):4759-4766.
[25]MU Panwei,JIANG Ping,WANG Manman,et al.Oestrogen exerts anti-inflammation via p38 MAPK/NF-κB cascade in adipocytes[J].Obesity Research&Clinical Practice,2016,10(6):633-641.
[26]Rom O,Avezov K,Aizenbud D,et al.Cigarette smoking and inflammation revisited[J].Respiratory Physiology&Neurobiology,2013,187(1):5-10.
[27]JIANG Junxia,ZHANG Shuijuan,SHEN Huijuan,et al.Rac1 signaling regulates cigarette smoke-induced inflammation in the lung via the Erk1/2 MAPK and STAT3 pathways[J].Biochimica et Biophysica ActaMolecular Basis of Disease,2017,1863(7):1778-1788.
[28]Nguyen T,Sherratt P J,Pickett C B.Regulatory mechanisms controlling gene expression mediated by the antioxidant response element[J].Annual Review of Pharmacology and Toxicology,2003,43:233-260.